Application of BAZ2B inhibitor in preparation of medicine for treating endometrial fibrosis or intrauterine adhesion

Abstract

The invention discloses an application of a BAZ2B inhibitor in preparation of a medicine for treating endometrial fibrosis or intrauterine adhesion. The BAZ2B inhibitor comprises an inhibitor for inhibiting and / or reducing activity and expression of BAZ2B, namely, the activity of a BAZ2B gene can be inhibited and / or reduced, and substances for inhibiting and / or reducing expression of the BAZ2B gene or inhibiting and / or reducing transcription and translation of the BAZ2B gene can be used as the BAZ2B inhibitor and can be used for treating endometrial fibrosis or intrauterine adhesion.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of a BAZ2B inhibitor in the preparation of a drug for treating endometrial fibrosis or intrauterine adhesions. Background Art

[0002] Intrauterine adhesions (IUA) are a major cause of reproductive health problems in women, with an increasing incidence, particularly among women of childbearing age. It has become the second most common reproductive health issue after polycystic ovary syndrome (PCOS). Clinical manifestations include decreased menstrual volume, amenorrhea, and subsequent infertility. Intrauterine adhesions impair endometrial receptivity in early pregnancy, leading to embryo implantation failure and recurrent miscarriage. In the second and third trimesters, IUAs are more susceptible to premature birth, abnormal placental attachment, peripartum hemorrhage, and poor uterine involution. Currently, the most commonly used treatment for moderate to severe IUAs is hysteroscopic IUA separation combined with postoperative estrogen-progestin therapy. However, the high recurrence rate and suboptimal reproductive outcomes make IUAs a difficult problem. The primary cause is endometrial scarring and destruction of the basal layer, leading to failure of endometrial regeneration, a problem that cannot be addressed or prevented through surgery or estrogen-progestin therapy. Therefore, clarifying the mechanisms that cause postoperative endometrial fibrosis after IUAs and identifying appropriate targets for intervention are of great scientific and clinical significance.

[0003] The specific molecular mechanisms underlying the formation of intrauterine adhesions remain unclear. Several unproven hypotheses exist, including the fibrosis-proliferation theory, the abnormal stem cell differentiation hypothesis, the dysregulated intrauterine inflammatory microenvironment hypothesis, and dysregulation of signaling pathways. Among these, fibrosis-proliferation is the most widely studied hypothesis, positing that the transformation of endometrial stromal cells into myofibroblasts is an early stage of endometrial fibrosis and a key factor in the development of intrauterine adhesions. Uterine curettage during pregnancy is considered a major contributing factor, followed by factors such as endometrial infection. Numerous studies have demonstrated that abnormal increases in cell growth factors and inflammatory factors are key factors in the development of endometrial stromal cell fibrosis. Transforming growth factor-β1 (TGF-β1) in particular can induce overactivation of endometrial stem cells (ESCs) and their differentiation into fibroblasts (MFs), activating multiple signaling pathways, including MAPK and S-mad, leading to abnormal secretion of collagen fibrillary proteins and abnormal endometrial proliferation and fibrosis.

[0004] BAZ2B is a component of the imitation-SWI chromatin remodeling complex. Studies have shown that ISWI family members participate in numerous biological processes within the cell nucleus, including gene expression regulation, DNA replication, and chromatin remodeling. BAZ2B possesses a classic bromodomain that recognizes histone H3K14ac acetylation, a modification that activates transcriptional activation of chromatin genes, suggesting that this gene may play a role in transcriptional regulation. Summary of the Invention

[0005] The application aims to provide an application of a BAZ2B inhibitor in preparing a drug for treating endometrial fibrosis or intrauterine adhesion.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the application is as follows:

[0007] In a first aspect, the application provides an application of a BAZ2B inhibitor in preparing a drug for treating endometrial fibrosis or intrauterine adhesion.

[0008] The BAZ2B inhibitor includes an inhibitor that can inhibit and / or reduce the activity and expression of BAZ2B, i.e. a substance that can inhibit and / or reduce the activity of the BAZ2B gene, inhibit and / or reduce the expression of the BAZ2B gene, or inhibit and / or reduce the transcription and translation of the BAZ2B gene, which can be used as a BAZ2B inhibitor and can be used for treating endometrial fibrosis or intrauterine adhesion.

[0009] The BAZ2B inhibitor is at least one of a small-molecule chemical inhibitor, a polypeptide, an antibody, or a biological material containing the small-molecule inhibitor, which has an inhibitory effect on the activity of BAZ2B.

[0010] The BAZ2B inhibitor is an expression vector containing an siRNA for knocking down the BAZ2B gene and an expression regulation sequence related to the operation, or a lipid nanoparticle loaded with an siRNA for knocking down the BAZ2B gene.

[0011] The siRNA sequence is shown in SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3.

[0012] The small-molecule chemical inhibitor having an inhibitory effect on the activity of BAZ2B is selected from GSK2801.

[0013] The chemical structure of GSK2801 (J Med Chem. 2016 Feb 25; 59(4): 1410-24.) is as follows:

[0014] Due to the adoption of the above technical scheme, the application has the following advantages and beneficial effects:

[0015] The application researches and finds that BAZ2B highly expressed by endometrial stromal cells in the pathogenesis of intrauterine adhesion can regulate the expression of extracellular matrix (ECM) related genes through an epigenetic mechanism, and promote the transformation of endometrial stromal cells to myofibroblast differentiation, thereby promoting endometrial fibrosis and damaging the regenerative capacity of the endometrium. Therefore, targeting BAZ2B has the effect of treating intrauterine adhesion or endometrial fibrosis. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Schematic diagram of the discovery that the epigenetic factor BAZ2B is a potential regulatory gene for endometrial fibrosis.

[0017] Figure 2 Schematic diagram of the results of the small molecule GSK2801 inhibiting the secretion of type I collagen in endometrial cells.

[0018] Figure 3 Schematic diagram of the results of the small molecule GSK2801 reversing endometrial fibrosis.

[0019] Figure 4 Schematic diagram showing elevated expression of BAZ2B in endometrial stromal cells of intrauterine adhesions.

[0020] Figure 5 Schematic diagram of BAZ2B regulating the proliferation and migration ability of endometrial stromal cells.

[0021] Figure 6 Schematic diagram of BAZ2B promoting the contractility of endometrial stromal cells.

[0022] Figure 7 Schematic diagram of BAZ2B promoting endometrial fibrosis.

[0023] Figure 8 Schematic diagram of GSK2801 inhibiting the progression of intrauterine adhesions in mice. DETAILED DESCRIPTION

[0024] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the following specific description is illustrative rather than restrictive and should not be used to limit the scope of protection of the present invention.

[0025] Example 1

[0026] The experimental model and method in the embodiment of the present invention are as follows:

[0027] 1. Establishment of a Human Endometrial Stromal Cell Fibrosis Model and Epigenetic Small Molecule Drug Screening

[0028] TGF-β1-induced endometrial fibrosis model: Human primary endometrial stromal cells were cultured to a density of approximately 60-70%, treated with 10 ng / ml TGF-β1 for 24 hours, and then treated with epigenetic small molecule drugs to a final concentration of 1 μM. After 48 hours, the cells were treated with trizol and subjected to subsequent real-time quantitative PCR (qRT-PCR) experiments.

[0029] 2. qRT-PCR detection of mRNA expression

[0030] RNA was extracted from cell and tissue samples and then reverse-transcribed into cDNA. qRT-PCR was performed using SYBR Green qPCR Master Mix (Hunan Aikerui Bioengineering Co., Ltd.), with cDNA and primers added according to the manufacturer's instructions. qRT-PCR reactions were performed on a QuantStudio 7 Real-Time PCR System (Thermo Fisher Scientific). All data were normalized using human actin B as an internal control.

[0031] 3. Cell Immunofluorescence Experiment

[0032] Human primary endometrial stromal cells were seeded on coverslips at a density of 40-50%, rinsed three times with ice-cold PBS for 5 minutes each time, fixed in a pH 7.2, 4% paraformaldehyde (0.1M PBS) solution for 10 minutes, rinsed three times with PBS for 5 minutes each time, permeabilized in a permeabilization reagent containing 0.3% TritonX-100 at room temperature for 15 minutes, blocked with 5% BSA at room temperature for 1 hour, incubated with primary antibody overnight, washed, incubated with secondary antibody at room temperature for 2 hours, incubated with DAPI for 15-20 minutes after washing, fixed with mounting medium (anti-fluorescence quenching fixative) after washing, and photographed and recorded with a confocal microscope.

[0033] 4. shRNA Knockdown and Plasmid Overexpression

[0034] The siRNAs used to knock down BAZ2A and BAZ2B were purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd., and the BAZ2B overexpression plasmid was purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd. (pRP[Exp]-CAG>hBAZ2B).

[0035] 5. Western Blot Detection

[0036] Total protein from cells and tissues was extracted using ice-cold RIPA buffer containing protease inhibitors (Shanghai Yazyme Biotechnology Co., Ltd.). Concentrations were determined using a BCA assay kit (Thermo Fisher Scientific) and normalized to the lowest concentration. Equal amounts of protein from different groups were electrophoresed on a 10% Tris-glycine SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk and incubated with a primary antibody overnight at 4°C. The membrane was washed three times with TBST for 5 minutes each, followed by incubation with a secondary antibody diluted 1:2000 at room temperature for 1 hour. The membrane was then washed three times with TBST for 8 minutes each, and then developed.

[0037] VI. Tissue Immunohistochemistry and Immunofluorescence Detection

[0038] After paraffin embedding, human endometrial tissue was deparaffinized using the following steps: xylene (10 minutes x 2) → anhydrous ethanol (5 minutes x 2) → graded ethanol (95% → 80% → 70%) (3 minutes each) → hydration (distilled water (5 minutes). Antigen retrieval: Sections were immersed in antigen retrieval solution (e.g., sodium citrate, pH 6.0) and microwaved on high for 10 minutes → cooled naturally → rinsed with PBS (3 x 5 minutes). Endogenous enzyme inactivation: Incubate with 3% H₂O₂ (methanol / PBS) at room temperature for 10-15 minutes → rinsed with PBS (3 x 5 minutes). Blocking: Add 5% BSA or normal serum dropwise and block at room temperature for 30 minutes → remove excess liquid. Primary antibody incubation: Add diluted primary antibody dropwise → Incubate at 4°C overnight or at room temperature for 1-2 hours (in a humidified chamber to prevent drying) → rinsed with PBS (3 x 5 minutes). Secondary antibody incubation: Add enzyme-conjugated secondary antibody (e.g., HRP-conjugated anti-rabbit IgG) dropwise → Incubate at room temperature for 30-60 minutes → rinsed with PBS (3 x 5 minutes). Color development: DAB color development (HRP system): Add freshly prepared DAB solution dropwise → Monitor under microscope (positive signal appears brownish-brown) → Stop color development in water. Counterstaining and mounting: Counterstain nuclei with hematoxylin for 1-2 minutes → Return to blue in running water → Dehydrate and clear (gradient ethanol → xylene → neutral gum mounting).

[0039] 7. Collagen Contraction Test

[0040] A rat collagen type I solution (1 mg / mL) was diluted with an appropriate buffer and then added to a suspension of primary human endometrial stromal cells. The volume ratio of the cell to collagen mixture was 1:3. The mixture was added to a culture plate and incubated in a 37°C incubator to allow the collagen matrix to solidify. After solidification, culture medium was added to overlay the collagen matrix. Cells were stimulated with fibrosis-related factors, such as TGF-β1, to enhance collagen synthesis and matrix contraction. Collagen matrix contraction was observed at designated time points (24 and 48 hours). Images were captured using a microscope, and the area change after matrix contraction was measured using ImageJ Pro 8.0 to quantitatively assess the cells' collagen synthesis and contraction capacity.

[0041] 8. Construction of Mouse Intrauterine Adhesion Model

[0042] Female mice (8-10 weeks old) were purchased from Shanghai Slake Laboratory Animal Co., Ltd. and housed in the animal facility of Tongji University's Hubei Campus. Animal ethics were approved by the Tongji University Experimental Animal Ethics Committee. The model was established as follows: During proestrus, mice were anesthetized with anesthetic. Laparotomy was performed on the back or abdomen to expose the uterine horns. The endometrial mucosa was scraped repeatedly with a microcurette, and the abdomen was sutured closed. Fourteen days after modeling, mice were sacrificed, and the uterus was removed. The uterine horns were assessed for swelling, deformation, and color change. The uterine cavity was examined for narrowing or occlusion, the presence of fibrous adhesions (adhesion bands), and the location, extent, and severity of adhesions. Masson staining, which specifically displays collagen fibers (blue), was used to quantitatively assess the extent of collagen deposition (fibrosis) in the endometrial stroma and adhesions. Immunohistochemistry was used to detect fibrosis markers, including α-SMA, myofibroblast markers, and type I collagen.

[0043] In the first step, epigenetic subtype drug screening identified BAZ2B as a potential regulator of endometrial fibrosis.

[0044] The most significant feature of intrauterine adhesions is that after artificial abortion and repeated curettage, due to excessive trauma to the endometrium and myometrium, especially in the case of concurrent infection, endometrial stromal cells differentiate into myofibroblasts and secrete a large amount of extracellular matrix (ECM), causing endometrial stromal cell fibrosis, which in turn leads to the failure of normal repair of endometrial tissue and the production of scar tissue.

[0045] In order to discover epigenes that inhibit the formation of endometrial fibrosis, the present invention uses human endometrial stromal cells to perform epigenetic drug screening. The specific process is as follows: Figure 1 As shown in A, Figure 1Schematic diagram of the discovery of the epigenetic factor BAZ2B as a potential regulatory gene for endometrial fibrosis. B is a schematic diagram of 218 small molecule drugs targeting epigenetic recognition protein domains, HDACs, histone acetyltransferases, histone demethylases, histone methyltransferases, and sirtuins. C is a schematic diagram of the discovery of 19 small molecules that inhibit the expression of Col1A1 (a collagen gene) and α-SMA (a fibrosis marker gene) among 218 small molecule drug screens. D is a schematic diagram of the discovery of the small molecule drug GSK2081 as a suppressor of α-SMA gene expression. E is a schematic diagram of the discovery of the small molecule drug GSK2081 as a suppressor of COL1A1 gene expression. F is a diagram of the validation of the BAZ2A knockdown efficiency of different siRNAs in human endometrial stromal cells. Quantitative PCR experiments revealed that BAZ2A siRNAs #2 and #3 were able to knock down BAZ2A expression. Significance is indicated by *P < 0.05. Figure G shows knockdown of BAZ2A using BAZ2A siRNA#3 in human endometrial stromal cells, which did not affect Col1A1 and α-SMA gene expression. Figure H shows the effectiveness of different siRNAs in knocking down BAZ2B in human endometrial stromal cells. Quantitative PCR revealed that BAZ2B siRNA#1 and 3 were able to knock down BAZ2B expression. Significance is indicated by *P < 0.05. Figure I shows knockdown of BAZ2B using BAZ2B siRNA#1 and 3 in human endometrial stromal cells, which significantly inhibited Col1A1 and α-SMA gene expression. Significance is indicated by *P < 0.05.

[0046] Figure 1 A is an epigenetic small molecule drug screening experiment. First, human endometrial stromal cells were cultured in a 12-well cell culture plate, and epigenetic small molecule drugs (218 small molecules) targeting epigenetic recognition protein domains, HDAC, histone acetyltransferase, histone demethylase, histone methyltransferase, Sirtuin, etc. were added ( Figure 1 The cells were treated with 19 small molecules (shown in Figure B) for 48 hours, and the expression of Col1A1 (collagen) and α-SMA (a marker gene for cell fibrosis) genes was detected by real-time quantitative PCR (qRT-PCR). It was found that 19 small molecules could significantly inhibit the expression of Col1A1 and α-SMA genes (*P<0.05) ( Figure 1 (Figure C). Endometrial stromal cells were further treated with TGF-β1 for 24 hours and 19 small molecules were added for 48 hours. The results showed that GSK2801 significantly inhibited the expression of Col1A1 and α-SMA genes ( Figure 1 The above results indicate that GSK2801 is an inhibitor of BAZ2A / B.

[0047] The chemical structure of GSK2801 (J Med Chem. 2016 Feb 25;59(4):1410-24.) is as follows:

[0048]

[0049] To further clarify whether it is BAZ2A or BAZ2B that regulates Col1A1 and α-SMA gene expression, three antisense oligonucleotide (siRNA) sequences for human BAZ2A were used to knock down BAZ2A, and three antisense oligonucleotide (siRNA) sequences for human BAZ2B were used to knock down BAZ2B. The results showed that knocking down BAZ2A did not affect the expression of Col1A1 and α-SMA genes. In contrast, knocking down BAZ2B significantly inhibited the expression of Col1A1 and α-SMA genes ( Figure 1 The above results indicate that BAZ2B is a potential epigenetic regulatory gene for intimal fibrosis.

[0050] The sequences of the three human BAZ2A antisense oligonucleotides (siRNA) are as follows:

[0051] siBAZ2A-1: 5'-GGCACCACUACAGAGAUAUTT-3' (SEQ ID NO. 1);

[0052] siBAZ2A-2: 5'-GGCAGAUGAUAGUCAAACATT-3' (SEQ ID NO. 2);

[0053] siBAZ2A-3: 5'-GGCCAGAAAUAAGCGGAAATT-3' (SEQ ID NO. 3).

[0054] The sequences of the three human BAZ2B antisense oligonucleotides (siRNA) are as follows:

[0055] siBAZ2B-1: 5'-GAGGCCGAGAAACGAAUAATT-3' (SEQ ID NO. 4);

[0056] siBAZ2B-2: 5'-GCGUAUUCCAUUGGAAUAUTT-3' (SEQ ID NO. 5);

[0057] siBAZ2B-3: 5'-GGGUGGUAGAAGGUAAACUTT-3' (SEQ ID NO. 6);

[0058] Figure 1 siNC is the control siRNA, with a random sequence as follows:

[0059] 5'-UCUACAGCGAUUCUCGUGCTT-3' (SEQ ID NO. 7).

[0060] In human intrauterine adhesion diseases, endometrial cell fibers have two significant characteristics: one is a significant increase in the secretion of collagen by cells, and the other is the transformation of endometrial stromal cells into myofibroblasts. Therefore, in order to prove that GSK2801 has an intervention effect on endometrial fibrosis, it should first be clarified that GSK2801 has an inhibitory effect on collagen production during the fibrosis process of endometrial stromal cells. The present invention used immunofluorescence experiments and found that the use of GSK2801 to treat endometrial stromal cells can significantly inhibit the expression of Col1A1. The results are as follows: Figure 2 As shown, Figure 2 Schematic diagram of the results of the small molecule GSK2801 inhibiting type I collagen secretion in endometrial cells. A shows the inhibition of type I collagen secretion (Col1A1) by GSK2801 in human endometrial stromal cells. Col1A1 is red fluorescent, and DAPI (4',6-diamidino-2-phenylindole), a nuclear fluorescent dye, is blue fluorescent. B shows the inhibition of type I collagen secretion (Col1A1) by GSK2801 in TGF-β1-induced myofibroblasts. As shown in the figure, GSK2801 inhibits type I collagen secretion (Col1A1) in endometrial stromal cells. Endometrial stromal cells were treated with TGF-β1 (endometrial fibrosis inducer) for 24 hours and then with GSK2801 for 48 hours. GSK2801 reversed TGF-β1-induced collagen production.

[0061] Figure 3Schematic diagram of the results of the small molecule GSK2801 reversing endometrial fibrosis. A is a schematic diagram showing TGF-β1 promoting the differentiation of endometrial stromal cells into myofibroblasts; B is a schematic diagram of immunofluorescence experiments demonstrating that GSK2801 inhibits α-SMA protein expression in endometrial stromal cells; phalloidin is green fluorescence, α-SMA is red fluorescence, and DAPI is blue fluorescence. C is a schematic diagram of immunofluorescence experiments showing that GSK2081 can reverse the TGF-β1-induced increase in α-SMA protein expression and bundling, indicating that GSK2801 can inhibit the differentiation of human endometrial stromal cells into myofibroblasts. As can be seen from the figure, the increased expression and bundling of α-SMA protein are significant features in the process of endometrial cell fibrosis. Treatment of HESC cells (human endometrial stromal cells) with GSK2801 can significantly inhibit the expression of α-SMA protein. HESC cells were treated with TGF-β1 for 24 hours and then treated with GSK2801 for 48 hours. It was found that GSK2801 can reverse the increased expression and bundling of α-SMA protein induced by TGF-β1 ( Figure 3 Thus, GSK2801 can reverse endometrial fibrosis ( Figure 3 (as shown in A in the figure).

[0062] The second step is to confirm that BAZ2B is a key regulatory gene for endometrial fibrosis

[0063] Figure 4Figure 1. Schematic diagram of elevated expression of BAZ2B in endometrial stroma cells in patients with intrauterine adhesion. A, RNA was extracted from endometrial samples obtained from patients with intrauterine adhesion and normal persons, and quantitative PCR was performed. The results showed that BAZ2A mRNA was slightly elevated in the endometrium of patients with intrauterine adhesion. B, RNA was extracted from endometrial samples obtained from patients with intrauterine adhesion and normal persons, and quantitative PCR was performed. The results showed that BAZ2B mRNA was significantly elevated in the endometrium of patients with intrauterine adhesion (P < 0.001 vs normal persons). C, Western blotting confirmed that BAZ2B was significantly elevated in the endometrium of patients with intrauterine adhesion. ACTB was used as an internal control. D, Immunohistochemical staining showed that BAZ2B was significantly elevated in the endometrial stroma cells of patients with intrauterine adhesion. E, UMAP plot of single-cell sequencing of endometrial samples from normal persons and patients with intrauterine adhesion. It can be seen from the figure that endometrial stromal cells account for the majority in the endometrium, and the number of differentiated myofibroblasts is small. F, Analysis of single-cell data from normal persons and patients with intrauterine adhesion showed that BAZ2B was expressed in endometrial cells from normal persons and patients with intrauterine adhesion. G, Analysis of mRNA data from normal persons and patients with intrauterine adhesion showed that BAZ2B was significantly elevated in activated myofibroblasts in patients with intrauterine adhesion (*P < 0.05 vs normal persons). H, Analysis of mRNA data from normal persons and patients with intrauterine adhesion showed that BAZ2B was not differentially expressed in myofibroblasts from normal persons and patients with intrauterine adhesion, indicating that BAZ2B drives the differentiation of activated endometrial fibroblasts into myofibroblasts. E-H, Public single-cell data (GSE215968) was used for analysis, and it was found that BAZ2B was significantly elevated in activated fibroblasts in patients with intrauterine adhesion (P < 0.05 vs normal persons). I, Immunofluorescence double staining showed that BAZ2B was highly expressed in activated endometrial stromal cells in patients with intrauterine adhesion. BAZ2B was red fluorescence, a-SMA was green fluorescence, and DAPI was blue fluorescence. J, Immunofluorescence staining showed that BAZ2B was highly expressed in endometrial stromal cells in patients with intrauterine adhesion. BAZ2B was green fluorescence, and DAPI was blue fluorescence.

[0064] Immunoblotting, qRT-PCR, and immunohistochemical staining were used to detect the expression levels of BAZ2B in normal endometrial tissues and intrauterine adhesion tissues (12 cases each), and it was found that BAZ2B in the endometrial tissues and endometrial stromal cells of patients with intrauterine adhesion was significantly higher than that in normal endometrial tissues and endometrial stromal cells. Figure 4A reanalysis of the single-cell endometrial tissue data (GSE215968) from patients with severe intrauterine adhesions and normal controls revealed that BAZ2B expression was significantly elevated in fibroblasts (endometrial stromal cells) from patients with intrauterine adhesions, compared to no increase in myofibroblasts from patients with intrauterine adhesions, suggesting that BAZ2B plays a role in promoting the activation of endometrial fibroblasts ( Figure 4 Tissue immunofluorescence double staining experiments revealed that BAZ2B expression was significantly increased in activated endometrial stromal cells of patients with intrauterine adhesions ( Figure 4 Primary endometrial stromal cells were further isolated from endometrial tissues of patients with intrauterine adhesions and normal subjects, and immunofluorescence assays revealed that the expression level of BAZ2B was significantly increased in the endometrial stromal cells of patients with intrauterine adhesions ( Figure 4 These results suggest that BAZ2B plays an important role in the activation of endometrial stromal cells and their differentiation into myofibroblasts during the development of intrauterine adhesions.

[0065] Compared with normal endometrial stromal cells, myofibroblasts have stronger migration, proliferation, and extracellular matrix secretion abilities. To further explore the specific regulatory mechanisms of BAZ2B on endometrial fibrosis, we used Transwell, EdU, and CCK-8 to detect the regulation of BAZ2B on the proliferation and migration of human endometrial stromal cells. Figure 5Figure 1 is a schematic diagram of BAZ2B regulating the proliferation and migration of endometrial stromal cells. Figure A is a schematic diagram of BAZ2B inhibition in in vitro cell experiments. As can be seen, siRNA was used to knock down BAZ2B in human endometrial stromal cells. Cell proliferation (EdU detection) experiments found that knocking down BAZ2B inhibited cell proliferation. EdU is green fluorescence, and DAPI is blue fluorescence. Figure B is a schematic diagram of BAZ2B overexpression in in vitro cell experiments. As can be seen, plasmids were used to overexpress the BAZ2B gene in human endometrial cells. Cell proliferation (EdU detection) experiments found that plasmid-overexpressed BAZ2B promoted cell proliferation. EdU is green fluorescence, and DAPI is blue fluorescence. C is a schematic diagram of BAZ2B inhibition in cell proliferation regulation in vitro. As shown in the figure, BAZ2B knockdown in human endometrial cells using siRNA was found to inhibit cell proliferation in CCK-8 assays. D is a schematic diagram of BAZ2B overexpression in vitro. As shown in the figure, BAZ2B overexpression in human endometrial cells using a plasmid was found to promote cell proliferation in CCK-8 assays. EdU and CCK8 assays confirmed that BAZ2B promoted the proliferation of human endometrial stromal cells. E is a schematic diagram of BAZ2B inhibition in cell migration in vitro. As shown in the figure, BAZ2B knockdown in human endometrial cells using siRNA was found to inhibit cell migration in Transwell assays. Figure F is a schematic diagram of in vitro cell experiments demonstrating the regulation of cell migration by overexpressing BAZ2B. As shown in the figure, plasmid-based overexpression of the BAZ2B gene in human endometrial cells promoted cell migration in Transwell assays. The present invention discovered that knocking down BAZ2B inhibited the migration and proliferation of human endometrial stromal cells. Conversely, overexpression of BAZ2B promoted their migration and proliferation, suggesting that BAZ2B promotes the differentiation of endometrial stromal cells into myofibroblasts.

[0066] During the fibrosis of endometrial adhesion cells, there is a dynamic and bidirectional interaction between cells and ECM, especially their mechanical interaction. The morphological characteristics of endometrial fibrosis are high expression of α-SMA, and the increase of α-SMA stress fibers promotes the cell's own contractility. To further clarify whether BAZ2B affects the contractility of cells, the present invention used collagen gel contractility experiments, and the results are as follows: Figure 6 As shown, Figure 6Schematic diagram of BAZ2B promoting endometrial stromal cell contractility. A is a schematic diagram showing the regulation of collagen contractility in endometrial stromal cells after BAZ2B inhibition in vitro; B is a schematic diagram showing the statistical differences in collagen contractility after BAZ2B knockdown using Image Pro analysis; the figure shows that BAZ2B knockdown in human endometrial stromal cells inhibits their contractility. C is a schematic diagram showing the regulation of collagen contractility in endometrial stromal cells after BAZ2B overexpression in vitro; D is a schematic diagram showing the statistical differences in collagen contractility after BAZ2B overexpression using Image Pro analysis; the figure shows that BAZ2B overexpression in human endometrial stromal cells promotes their contractility.

[0067] Figure 7 Schematic diagram of BAZ2B promoting endometrial fibrosis. A is a cell immunofluorescence assay demonstrating that BAZ2B knockdown inhibits α-SMA protein expression in endometrial stromal cells. The figure shows that BAZ2B knockdown in human endometrial stromal cells reverses TGF-β1-induced increases in α-SMA protein expression and bundling, indicating that BAZ2B inhibition can inhibit myofibroblast differentiation. Phalloidin is shown as green fluorescence, α-SMA as red fluorescence, and DAPI as blue fluorescence. B is a cell immunofluorescence assay demonstrating that BAZ2B knockdown inhibits type I collagen secretion in endometrial stromal cells. The figure shows that BAZ2B knockdown in TGF-β1-induced myofibroblasts inhibits type I collagen secretion (Col1A1), indicating that BAZ2B inhibition can inhibit type I collagen secretion in human myofibroblasts. DAPI (4',6-diamidino-2-phenylindole) is shown as blue fluorescence. C is a schematic diagram of a cell-based immunofluorescence assay demonstrating that overexpression of BAZ2B inhibits α-SMA protein expression in endometrial stromal cells. As shown in the figure, overexpression of BAZ2B in human endometrial stromal cells promotes TGF-β1-induced elevation of α-SMA protein expression and its bundling, indicating that BAZ2B promotes myofibroblast differentiation. D is a schematic diagram of a cell-based immunofluorescence assay demonstrating that overexpression of BAZ2B inhibits type I collagen secretion in endometrial stromal cells. As shown in the figure, overexpression of BAZ2B in TGF-β1-induced myofibroblasts promotes type I collagen secretion (Col1A1), indicating that inhibition of BAZ2B can inhibit type I collagen secretion in human myofibroblasts. E is a schematic diagram demonstrating that knockdown of BAZ2B reverses TGF-β1-induced myofibroblast differentiation in endometrial stromal cells. As shown in the figure, inhibition of BAZ2B expression can reverse endometrial fibrosis.

[0068] The expression of type I collagen and a-SMA protein in endometrial stromal cells is significantly increased and bundled, and the use of BAZ2B siRNA to treat HESC cells can significantly inhibit the expression of type I collagen and a-SMA protein, and conversely, overexpression of BAZB can promote the expression of type I collagen and a-SMA protein. After treating HESC cells with TGF-β1 for 24 hours, and then adding BAZ2B siRNA for 48 hours, it is found that knocking down BAZ2B can reverse the increase in expression of type I collagen and a-SMA protein and bundling induced by TGF-β1 Figure 7 , and conversely, overexpression of BAZB can promote the expression of type I collagen and a-SMA protein and bundling Figure 7 . Therefore, through the above experiments, it can be confirmed that BAZ2B is an early driver of endometrial fibrosis, which initiates the fibrosis process of endometrial stromal cells (TGF-β1), and inhibiting the expression of BAZ2B can reverse endometrial fibrosis Figure 7 .

[0069] Third, it is confirmed that GSK2801 can inhibit the progression of mouse adhesions disease

[0070] Confirm the estrus cycle of 8-10 week old C57BL / 6 female mice by vaginal smear, and perform subsequent modeling on estrus animals. The mechanical damage model modeling steps are as follows: the mice were fasted for 12 hours before the operation, but not deprived of water, first anesthetize the C57BL / 6 mice with anesthetic, make an incision in the lower abdomen of the mouse, expose the uterus, make an incision about 1 mm below the ovary, use a self-made mouse uterine curette to perform uterine curettage on the mouse through the incision below the cervix, stop curettage when the four walls of the uterine cavity appear rough, rinse the abdominal cavity with normal saline, suture the abdominal wall of the mouse, and the next day inject GSK2801 small molecule drug (10 mg / kg), inject once a day, for 13 consecutive days, and sacrifice the mice on the 15th day Figure 8 , take out the uterine paraffin-embedded sections, then HE stain and Masson stain, and then measure the endometrial thickness and calculate the number of glands. The results show that GSK2801 can significantly inhibit the progression of adhesion disease Figure 8 , as shown in FIGS. 8B-8E.

[0071] Figure 8Schematic diagram of GSK2801 inhibiting the progression of intrauterine adhesions in mice. A shows the timing and dosage of intraperitoneal injections of GSK2801 in mice with an intrauterine adhesion model. As can be seen, continuous intraperitoneal injections of GSK2801 resulted in no mortality or weight loss, demonstrating that the GSK2801 dosage is appropriate. B shows the uterine morphology of wild-type mice, the intrauterine adhesion model group, and the GSK2801-treated group after modeling. Morphological observation reveals that the uterus of mice with intrauterine adhesions is significantly narrowed and pale, while the uterine narrowing is significantly improved in the GSK2801-treated group, with increased uterine vascularity. C shows HE staining demonstrating that GSK2801 promotes endometrial repair in mice with intrauterine adhesions. Compared to the intrauterine adhesion group, the GSK2801-treated group showed intact epithelial cells, increased endometrial thickness, and structural integrity, demonstrating that GSK2801 promotes endometrial regeneration. D is a statistical diagram showing the effect of GSK2801 on the number of glandular formation in mice with intrauterine adhesions; this diagram demonstrates that GSK2801 promotes endometrial gland regeneration in mice. E is a diagram showing Masson staining demonstrating that GSK2801 inhibits the progression of endometrial fibrosis in mice with intrauterine adhesions; this diagram demonstrates that GSK2801 can protect against endometrial fibrosis in mice with intrauterine adhesions. F is a statistical diagram showing the effect of GSK2801 on the thickness of the endometrium in mice with intrauterine adhesions; this diagram demonstrates that GSK2801 increases endometrial thickness in mice with intrauterine adhesions, promoting endometrial regeneration in mice with intrauterine adhesions. G is a schematic diagram of the endometrial histochemistry of mice in the wild-type group, intrauterine adhesion model group, and GSK2801-treated group after modeling. As can be seen from the figure, compared with the intrauterine adhesion group, the expression of key markers of endometrial fibrosis α-SMA, Col1A1, and FN1 in the GSK2801 drug-treated group was significantly reduced, indicating that GSK2801 inhibits the differentiation of endometrial stromal cells into myofibroblasts and the deposition of extracellular matrix, thereby inhibiting the progression of endometrial fibrosis in intrauterine adhesions.

[0072] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with the present invention can make slight changes or modifications to equivalent embodiments using the above-mentioned technical contents without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the solution of the present invention.

Claims

1. Use of a BAZ2B inhibitor in the preparation of a medicament for treating endometrial fibrosis or intrauterine adhesions.

2. Use of the BAZ2B inhibitor according to claim 1 in the preparation of a medicament for treating endometrial fibrosis or intrauterine adhesions, characterized in that: The BAZ2B inhibitors include inhibitors that inhibit and / or reduce the activity and expression of BAZ2B, that is, substances that can inhibit and / or reduce the activity of the BAZ2B gene, inhibit and / or reduce the expression of the BAZ2B gene, or inhibit and / or reduce the transcription and translation of the BAZ2B gene can all be used as BAZ2B inhibitors and can be used to treat endometrial fibrosis or intrauterine adhesions.

3. Use of the BAZ2B inhibitor according to claim 1 in the preparation of a medicament for treating endometrial fibrosis or intrauterine adhesions, characterized in that: The BAZ2B inhibitor is at least one of a small molecule chemical inhibitor, a polypeptide, an antibody, and a biological material containing the small molecule inhibitor that has an inhibitory effect on BAZ2B activity.

4. Use of the BAZ2B inhibitor according to claim 1 in the preparation of a medicament for treating endometrial fibrosis or intrauterine adhesions, characterized in that: The BAZ2B inhibitor is an expression vector containing siRNA for knocking down the BAZ2B gene and an expression regulatory sequence related to operability, or lipid nanoparticles encapsulating siRNA for knocking down the BAZ2B gene.

5. Use of the BAZ2B inhibitor according to claim 4 in the preparation of a medicament for treating endometrial fibrosis or intrauterine adhesions, characterized in that: The siRNA sequences are shown in SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO.

3.

6. Use of the BAZ2B inhibitor according to claim 3 in the preparation of a medicament for treating endometrial fibrosis or intrauterine adhesions, characterized in that: The small molecule chemical inhibitor having an inhibitory effect on BAZ2B activity is selected from GSK2801, and the chemical structure of GSK2801 is as follows: 。

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